1. Field of the Invention
This invention relates to an improved method for producing polypeptides by bacterial fermentation. More specifically, this invention addresses the newly found problem of dissolved oxygen instability that is particularly manifest in large-scale bacterial fermentations.
2. Description of Related Art
The production of large quantities of relatively pure, biologically active polypeptides and proteins is important economically for the manufacture of human and animal pharmaceutical formulations, enzymes, and other specialty chemicals. For production of many proteins, recombinant DNA techniques have become the method of choice because large quantities of exogenous proteins can be expressed in bacteria and other host cells free of other contaminating proteins. The expression of proteins by recombinant DNA techniques for the production of cells or cell parts that function as biocatalysts is also an important application.
Producing recombinant protein involves transfecting host cells with DNA encoding the protein and growing the cells under conditions favoring expression of the recombinant protein. The prokaryote E. coli is favored as host because it can be made to produce recombinant proteins in high yields. Numerous U.S. patents on general bacterial expression of DNA encoding proteins exist, including U.S. Pat. No. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and non-bacterial gene; U.S. Pat. No. 4,673,641 on co-production of a foreign polypeptide with an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide such as IGF-I; U.S. Pat. No. 4,795,706 on expression control sequences to include with a foreign protein; and U.S. Pat. No. 4,710,473 on specific circular DNA plasmids such as those encoding IGF-I.
The electron transfer chain of bacterial organisms is capable of transferring electrons from substrates to molecular oxygen. The cytochromes are a group of iron-containing electron-transferring proteins on the electron transfer chain that act sequentially to transfer electrons from flavoproteins to molecular oxygen. The terminal cytochrome of the electron transfer chain is called cytochrome oxidase.
When a portion of a bacterial fermentor experiences a low dissolved oxygen (DO.sub.2) concentration, the bacterial organism is induced for the production of the cytochrome d oxidase complex. Fu et al., Mol. Gen. Genetics, 226:209-213 (1991). This complex has a higher affinity for oxygen than the cytochrome o oxidass complex normally used. Anraku and Gennis, TIBS, 12:262-266 (1987). Thus, when it is present, the cytochrome d complex allows the organism to continue with aerobic respiration under conditions of low oxygen concentration.
Ubiquinone is the electron transport mediator of the electron transport chain that is normally used by bacteria during vigorous aerobic growth. However, under conditions loosely defined as the approach of stationary phase [Poole and Ingledew, "Pathways of Electrons to Oxygen," in Neidhardt FC et al. (eds.) Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, Vol. 1 (American Society for Microbiology, Washington, D.C., 1987) p. 180], the production of an alternative electron transport mediator, menaquinone, is induced. In most high-cell-density fermentations, a large portion of the procedure is conducted with the cells in a condition approximating stationary phase or the approach thereof. This may be caused by limitation of the carbon/energy source to avoid oxygen depletion, by measures used to induce product formation, or by the presence of the product.
The cytochrome d oxidase complex has a higher affinity for menaquinone than for ubiquinone (Anraku and Gennis, supra), and presumably uses menaquinone as its principal electron transport mediator when menaquinone is present. Unfortunately, organisms using menaquinone as their electron transport mediator grow with a much reduced efficiency. The aerobic growth yield of organisms missing ubiquinone is about 30% that of a wild-type organism. Wallace and Young, Biochim. Biophys. Acta, 461:84-100 (1977).
Examples of microbial organisms with mutated electron transport mediators such as cytochrome d oxidase or cytochrome o oxidase are reported by Oden et al., Gene, 96:29-36 (1990); Oden and Gennis, J. Bacteriol., 173:6174-6183 (1991); Kelly et al., J. Bacteriol., 172:6010-6019 (1990); Iuchi et al., J. Bacteriol., 172:6020-6025 (1990); Soberon et al., J. Bacteriol., 172:1676-1680 (1990); Poole et al., J. Gen. Microbiol., 135:1865-1874 (1989); Fang et al., J. Biol. Chem., 264:8026-8032 (1989); James et al., FEMS Microbiol. Lett., 58:277-281 (1989); Green et al., J. Biol. Chem., 263:13138-13143 (1988); Soberon et al., J. Bacteriol., 171:465-472 (1989); Sharma et al., Indian J. Microbiol., 27:26-31 (1987); Yang, Arch. Microbiol., 144:228-232 (1986); Matsushita and Kaback, Biochemistry, 25:2321-2327 (1986); Tamura-Lis and Webster, Arch. Biochem. Biophys., 244:285-291 (1986); Au et al., J. Bacteriol., 161:123-127 (1985); McInerney et al., Eur. J. Biochem., 141:447-452 (1984); Lorence et al., J. Bacteriol., 157:115-121 (1984); Kranz et al., J. Bacteriol., 156: 115-121 (1983); Green and Gennis, J. Bacteriol., 154:1269-1275 (1983); Bryan and Kwan, Antimicrob. Agents Chemother., 19:958-964 (1981); Willison et al., FEMS Microbiol. Lett., 10:249-255 (1981); Willison and Haddock, FEMS Microbiol. Lett., 10:53-57 (1981); Hoffman et al., Eur. J. Biochem., 105:177-185 (1980); Hoffman et al., Eur. J. Biochem., 100:19-27 (1979); Haddock and Schairer, Eur. J. Biochem., 35:34-45 (1973); Haltia et al., EMBO J., 8:3571-3579 (1989); Dikshit et al., Arch. Biochem. Biophys., 293:241-245 (1992); Lemieux et al., J. Biol. Chem., 267:2105-2113 (1992); Minagawa et al., J. Biol. Chem., 267:2096-2104 (1992); Dassa et al., Mol. Gen. Genet., 229:341-352 (1991); Williams et al., Biochem. J., 276:555-557 (1991); Puustinen et al., Biochemistry, 30:3936-3942 (1991); Denis et al., J. Biol. Chem., 265:18095-18097 (1990); Chepuri et al., Biochim. Biophys. Acta, 1018:124-127 (1990); Andersson and Roth, J. Bacteriol., 171:6734-6739 (1989); Puustinen et al., FEBS Lett., 249:163-167 (1989); Daldal, J. Bacteriol., 170:2388-2391 (1988); Poole and Williams, FEBS Lett., 231:243-246 (1988); Georgiou et al., J. Bacteriol., 169:2107-2112 (1987); O'Brian and Maier, J Bacteriol , 161:507-514 (1985); Green et al., J. Biol. Chem., 259:7994-7997 (1984); Au et al., J. Bacteriol., 157:122-125 (1984); Matsushita et al., Proc. Natl. Acad. Sci. USA, 80:4889-4993 (1983); Sasarman, Rev. Can. Biol., 31:317-319 (1972); Van der Oost et al., EMBO J., 11:3209-3217 (1992); Deutch, 92nd General Meeting of the American Society for Microbiology, New Orleans, La., May 26-30, 1992, Abstr. Gen. Meet. Am. Soc. Microbiol., 92:272 (1992); Mogi et al., Biophys. J., 61:A284 (1992); Tron and Lemesle-Meunier, Curr. Genet., 18:413-420 (1990); Shioi et al., J. Bacteriol., 170:5507-5511 (1988); Oden and Gennis, J. Cell. Biol., 107:624A (1988); Webster and Georgiou, Fed. Proc., 44:abstract 678 (1985); Terriere et al., Biochem. Biophys. Res. Commun., 111:830-839 (1983); Green and Gennis, Fed. Proc., 41:abstract 3652 (1982); Tamura-Lis and Webster, Fed. Proc., 41:abstract 2799 (1982); Green et al., Fed. Proc,, 40:1669 (1981); Willison and John, J. Gen. Microbiol., 115: 443-450 (1979); and Hashimoto and Hino, J. Sci. Hiroshima Univ. Ser. B Div. 2 (Bot), 15:103-114 (1975).
Fermentors for culturing bacteria are normally agitated to transfer oxygen from the gaseous phase to the liquid phase (i.e., the medium), and, secondarily, to maintain uniform concentrations of medium components, including DO.sub.2, throughout the fermentor tank. The present invention is based on the unexpected finding that while during small-scale culturing to achieve DNA expression in bacteria the fermentation medium has a relatively stable DO.sub.2 concentration, during large-scale bacterial fermentations sudden and dramatic, essentially uncontrollable changes in the DO.sub.2 concentration of the medium may be experienced. These events prevent the successful and reproducible progression of large-scale fermentations, rendering them unsuitable for the production of high-quality protein or other products. When cultures are grown under glucose or other carbon/energy source limitation, feed control of the carbon/energy source can be programmed to provide adequate DO.sub.2 concentration under most circumstances; however, in large reactors, this control action is found by the present invention not to be sufficient to counteract precipitous biological events.
Accordingly, it is an object of this invention to provide an effective and reliable method for avoiding the problem of DO.sub.2 instabilities newly observed in bacterial fermentations.
It is a particular object to attain successful and reproducible progression of bacterial fermentations, especially large-scale bacterial fermentations, to produce high-quality polypeptides in an improved good manufacturing practice (GMP) process for FDA approval, and/or to produce the polypeptides in higher yield.
It is another object to provide a method for determining a culture's propensity for DO.sub.2 instability during the course of a bacterial fermentation.
These and other objects will be apparent to those of ordinary skill in the art.